整合子介导产生超广谱β-内酰胺酶志贺菌的多重耐药研究

Study of multi-drug resistance in producing extended spectrum-lactamases shigella mediated by integron

目的探讨产生超广谱β-内酰胺酶(ESBL)志贺菌的耐药特性及其耐药机制。方法采用K-B法药敏试验筛选可疑产ESBL志贺菌株;通过改良三维试验、E-test试验及相对水解率测定对可疑产ESBL志贺菌进行鉴定;采用TEM、SHV、CTX-M-1组、CTX-M-2组和CTX-M-9组β-内酰胺酶通用引物进行PCR检测,并对TEM和CTX-M-9组全编码基因引物等PCR扩增产物进行DNA序列分析;对产ESBL志贺菌进行结合传递试验,供体菌和接合子用稀释法进行MIC测定。对ESBL菌株进行Ⅰ类、Ⅱ类和Ⅲ类整合子的多重PCR检测,Ⅰ类整合子可变区PCR扩增产物进行DNA测序,确定耐药基因盒的种类和数量。结果275株志贺菌中有12株为产ESBL志贺菌,其基因型为CTX-M-14和CTX-M-1组;产ESBL志贺菌结合传递试验全部阳性,其结合子只对β-内酰胺类抗生素耐药。12株ESBL志贺菌只含Ⅰ类整合子,整合子可变区含有dfrA17-aadA5耐药基因盒。结论济南地区志贺菌对β-内酰胺类抗生素的交叉耐药由质粒介导,对磺胺类和氨基糖苷类多重耐药由整合子介导。

Objective To explore the characteristic and drug-resistance mechanism of ESBLs producing shigella.Methods The suspicious ESBLs producing isolates were screeninged by K-B disc diffusion method and they were confirmed by modified three-dimensional method,E-test and relative hydrolysis rate experiment.The conjugation experiment was performed to determine whether the resistance was transferable.Minimal inhibitory concentration（MIC） was detected with double agar dilution method.The partial blagene of these isolates were detected by PCR using universal primers for TEM,SHV,CTX-M-1 group,CTX-M-2 group,CTX-M-9 group,respectively.The entire blaCTX-M-9,blaTEM and others were amplified by PCR using the primers outside the open reading frame（ORF） of these β-lactamases and products were directly sequenced.The class Ⅰ,Ⅱ,Ⅲ integrons were identified with multi-PCR,and the PCR products of variable region of integronⅠ was sequenced.Results Of the 275 isolates,12 were identified as ESBLs producers.The genotypes of ESBLs were CTX-M-14 and CTX-M-3.All ESBLs producing isolates are positive to plasmid conjugative transfer test,the transconjugants are only resistance to beta-lactams.All 12 ESBL producing isolates contained intgronⅠ,which had the dfrA17-aadA5 gene cassettee on the variable region.Conclusion In Jinan area,shigella resisting to beta-lactams is mediated by plasmid,and multi-resisting to sulfanilamides and aminoglycosides mediated by integron.